Human Immunodeficiency Virus Type 1 (HIV-1) is the etiologic agent that is responsible for AIDS, a syndrome characterized by depletion of CD4+ T-lymphocytes and collapse of the immune system. HIV-1 infection is pandemic and HIV-associated diseases have become a world-wide health problem. Upon infection, HIV-1 integrates into the cellular genome of an infected cell. HIV-1 infection then leads to two different scenarios: productive infection and latent infection. Productive infection occurs most frequently and leads to death of the infected cell after release of progeny virus. During latent infection, which is rare, HIV-1 genes are not expressed after proviral integration, resulting in an infected cell that is characterized by transcriptionally silent HIV-1 genes. These fully replication-competent HIV-1 can persist dormant in cells for several years and then become reactivated (Chun et al, 1995, Nature Med 1(12):1284-1290; Chun et al., 1997, Proc Natl Acad Sci USA 94(24):13193-13197; for review, see Bisgrove, 2005, Expert Rev Anti Infect Ther 3(5):805-814).
Current treatments of AIDS typically seek to block one or more steps involved in the production of viral particles. Treatment options involve administration of reverse transcriptase inhibitors, inhibitors of viral protease, fusion, entry, or integration inhibitors in different combinations to block multiple steps in the viral life cycle. This approach, termed highly active antiviral therapy (HAART) has greatly decreased morbidity and mortality in people infected with HIV-1 (Palella et al., 1998, N Engl J Med 338(13):855-860).
However, long-term follow-up studies have shown that HAART alone is not effective in completely eliminating HIV-1 in infected patients. In most cases, upon ceasing HAART, a rapid rebound in viremia occurs even after years of successful treatment with undetectable viral loads (Davey et al, 1999, Proc Natl Acad Sci USA 96(26):15109-15114; Cohen and Fauci, 2001, Adv Intern Med 46:207-246). The rebound in viremia is believed to be due at least in part to the reactivation of latent HIV-1. Latent forms of are not sensitive to HAART because these drugs (e.g., reverse transcriptase inhibitors, viral protease inhibitors) are only active against actively replicating forms of HIV-1. Although the frequency of latently-infected cells is only about 0.03-3 infectious units per million resting CD4+T-cells (Siliciano et al., 2003, Nature Med 9(6):727-728), this latent population of HIV-1 serves as a source of virus for reseeding the infection after discontinuation of HAART. Due to the longevity of this latent HIV-1 reservoir, it is unlikely that HAART alone can ever clear it completely (Siliciano et al., ibid).
HIV-1 latency is closely tied to expression of HIV-1 genes, i.e., to HIV-1 transcription, which initiates at a promoter located in the 5′ LTR driving transcription of the viral genome. The LTR comprises essentially 4 regions: a negative regulatory element (NRE), an enhancer region, a core promoter and a 5′ untranslated region (UTR) (for review, see Bisgrove, 2005, Expert Rev Anti Infect Ther 3(5):805-814). Of particular interest for reactivation of HIV-1 expression is the enhancer region, which can be subdivided into a distal and proximal region. Several transcription factors bind to these regions. For example, Ets-1 and LEF-1 bind to the distal enhancer region, while the inducible transcription factors nuclear factor-kappa B (NF-κB) and NF-AT bind to and reactivate HIV-1 transcription from the proximal enhancer.
Select viral proteins are also involved in reactivation of HIV-1 gene transcription. For example, one of the early proteins expressed from the HIV-1 genome is Tat, a viral transactivator that binds to an RNA recognition element (TAR) present in all viral transcripts and primarily drives high level of HIV-1 expression by enhancing transcriptional elongation by RNA polymerase II after binding to the HIV-1 LTR.
Recently, several lines of evidence pointed to an inhibitory effect of chromatin on HIV-1 gene expression initiated on the integrated HIV-1 genome. With respect to histone H3, a protein component of a nucleosome (the base unit of chromatin), acetylation or methylation of amino acid residue lysine 9 has been implicated in transcriptionally active or inactive chromatin, respectively. It has been recognized that nucleosomes can negatively regulate gene expression by, e.g., preventing access to the DNA binding sites of transcription factors, thereby reducing or silencing expression of nearby genes (Owen-Hughes and Workman, 1994, Crit Rev Eukaryot Gene Expr 4(4):403-441; Knezeetic and Luse, 1986, Cell 45(1):95-104).
Prior to transcriptional reactivation, 5 nucleosomes are precisely positioned in the 5′ LTR of HIV-1. Nucleosome nuc-0, encompassing part of the NRE region is separated from nucleosome nuc-1 by a 265 by nucleosome-free region, containing binding sites for transcription factors C/EBP, LEF-1, NF-κB, NF-AT, Sp1 and the TATA box (Verdin et al., 1993, EMBO J 12(12):4900; Jones and Peterlin, 1994, Anna Rev Biochem 63:717-743). Upon reactivation, nuc-1 is rapidly remodeled which may relieve a block to HIV-1 gene transcription. Reactivation of HIV-1 latency seems also to involve recruitment of acetyltransferase to the HIV-1 LTR, followed by acetylation of histones H3 and H4 (Lusic et al., 2003, EMBO J 22(24):6550-6561; Bisgrove, 2005, Expert Rev Anti Infect Ther 3(5):805-814). Thus, chromatin is an integral component of the HIV-1 transcriptional regulatory machinery and modulation thereof is expected to have a direct impact on the expression of HIV-1 genes.
Further, HIV-1 latency may also be explained by integration of the HIV-1 genome into heterochromatin, a transcriptionally repressive form of chromatin, that eventually may become reorganized leading to the reactivation of latent HIV-1 expression (Jordan et al., 2003, EMBO J 22(8):1868-1877). Another mechanism underlying HIV-1 latency may be transcriptional interference with a nearby gene (Han et al., 2004, J Virol 78(12):6122-6133).
Two strategies have been proposed to overcome the problem that current HAART is unable to completely clear the latent HIV-1 reservoir. The first one can be described as an intensified HAART aiming to prevent even a very low level of viral replication (Ramratnam et al., 2004, J Acquir Immune Defic Syndr 35(1):33-37). A second approach aims at eliminating the pool of latently infected cells by inducing HIV-1 replication in these cells, while maintaining the patient on HAART to prevent a spreading infection. The latently-infected cells would then be eliminated by the immune system or virus-mediated cell lysis.
In pursuing the second approach, purging the latent HIV-1 pool by reactivation of viral transcription, several clinical trials have been performed, although, with limited success so far. For example, studies using IL-2 or IL-2 and OKT3 have not shown significant reduction in the latent reservoir and viral rebound continues after cessation of HAART (Chun et al., 1999, Nat Med 5:651-655; van Praag et al., 2001, J Clin Immunol 21:218-226; Blankson et al., 2002, Ann Rev Med 53:557-593). Another potential drug useful for viral purging is IL-7 (Smithgall et al., 1996, J Immunol 156(6):2324-2330; Scripture-Adams et al., 2002, J Virol 76(24):13077-13082).
Recently, prostratin and the related 12-deoxyphorbol 13-phenylacetate (DPP) were described as promising inducers of latent HIV-1. Prostratin is a nontumor-promoting phorbol ester initially isolated in screens for inhibitors of HIV-1 replication (Gustafson et al., 1992, J Med Chem 35(11):1978-1986). However, further studies indicated that in addition to blocking HIV-1 infection, prostratin treatment also upregulated HIV-1 transcription from latent proviruses (Kulkosky et al., 2001, Blood 98(10:3006-15; Korin et al., 2002, J Virol 76(16):8118-8123; Biancotto et al., 2004, J Virol 78(19):10507-10515).
To be clinically useful, reactivators of latent HIV-1 expression must exhibit relatively low toxicity, permitting patients to withstand treatment with these agents (Perelson et al., 1997, Nature 387, 188-191). Although prostratin functions as a reactivator of latent HIV-1 expression and was observed to lack toxicity when applied for short time courses, in its current dosage regimen, prostratin may not be useful for long-term, multiround treatments in humans. Prostratin was reported to induce substantial growth arrest and cell death if administered in a concentration of >500 nM for more than 2 days (Williams et al., 2004, J Biol Chem 279(40):42008-42017). Thus, if prostratin is to be considered as a human therapeutic, it is unlikely that high-dose or protracted treatment will be tolerated. Consequently, either short-term and/or low-dose treatments will probably be the only alternative, since sustained administration of prostrating at a high-dose will probably result in dramatically negative side effects (Williams et at, 2004, J Biol Chem 279(40):42008-42017). However, no such protocols are available yet.
Histone acetylases and deacetylases play a major role in the control of gene expression. They regulate gene expression by acetylating and deacetylating lysine residues on histones as well as various transcription factors. The balance between the activities of histone acetylases, usually called acetyl transferases (HATs), and deacetylases (HDACs) determines the level of histone acetylation. Acetylated histones are associated with a relaxed, more open form of chromatin and activation of gene transcription, whereas deacetylated chromatin is associated with a more compacted form of chromatin and diminished transcription. Eleven different HDACs have been cloned from vertebrate organisms. Class I HDACs includes HDAC1, HDAC2, HDAC3, and HDAC8 (Van den Wyngaert et al., 2000, FEBS Lett 468:77-83). Class II HDACs includes HDAC4, HDAC5, HDAC6, HDAC7, HDAC7, HDAC9, and HDAC10 (Kao et al., 2000, Genes Dev 14:55-60; Grozinger et at, 1999, Proc Natl Acad Sci USA, 96:4868-73; Zhou et al., 2001, Proc Natl Acad Sci USA, 98:10572-77; Tong et al., 2002, Nucleic Acids Res 30:1114-23). HDAC11 has not been classified yet (Gao et al., 2002, J Biol Chem 277:25748-55). All share homology in their catalytic regions.
HDACs have also been implicated in the inhibition of HIV-1 gene expression and thus, may contribute to establishing or maintaining HIV-1 latency (Ylisastigui et al., 2004, AIDS 18(8):1101-1108). Further, it has been shown that NF-κB p50-HDAC1 complexes constitutively bind the latent HIV-1 LTR and induce histone deacetylation and repressive changes in chromatin structure of the HIV-1 LTR, changes that impair recruitment of RNA polymerase II and transcriptional initiation (Williams et al., 2006, EMBO J 25:139-149).
Thus, histone deacetylase (HDAC) inhibitors are also being considered as an adjuvant with HAART (see, Bisgrove, 2005, Expert Rev Anti Infect Ther 3(5):805-814). HDAC inhibitors have the ability to reactivate a range of HIV-1 subtypes in a variety of different cell types (Van Lint at al., 1996, EMBO J 15(5):1112-1120; Quivy et al., 2002, J Virol 76(21):11091-11103). Some HDAC inhibitors are already in clinical use for other purposes. For example, valproic acid is widely used to reduce epileptic seizures, and phenylbutyrate is used to treat sickle cell anemia and various forms of thalassemia, establishing their safety profile. Recently, it was suggested that the HDAC inhibitor valproic acid may have effects on the reactivation of latent HIV-1 (Ylisastigui et al., 2004, AIDS 18(8):1101-1108).
TSA, e.g., has been shown to inhibit HDAC1, leading to the recruitment of RNA polymerase to the latent HIV-1 LTR. This bound polymerase complex, however, remains non-processive, generating only short viral transcripts. Synthesis of full-length viral transcripts can be rescued by the expression of Tat (Williams et al, 2006, EMBO J 25:139-149).
Recently, Williams and Greene described compositions and methods for reactivating latent HIV-1 expression wherein they contacted a cell having an integrated HIV-1 genome with an reactivator of latent HIV-1 expression and with an inhibitor of an HDAC (U.S. Pat. No. 8,247,613).
Despite this progress cells latently infected with HIV-1 still represent an insurmountable barrier to viral eradication in infected patients. New approaches for the elimination of the latently infected HIV-1 cells are urgently needed (see Pomerantz, 2002, Curr Opin Invest Drugs 3:1133-1137). In view of this unfulfilled need, Applicants asked the question: “Can gene expression fluctuations, or ‘noise,’ be used as a drug discovery tool?” Studies to date hint that the answer might be yes. Some sources and phenotypic implications of gene expression noise have been investigated (Kaern et al., 2005, Nat Rev Genet 6:451-464; Balazsi and van Oudenaarden, 2011, Cell 144:910-925). On one hand, noise has been exploited as a probe to elucidate underlying structure-function relationships of genetic circuitry (Blake et al., 2003, Nature 422:633-637; Rosenfeld et al., 2005, Science 307:1962-1965; Austin et al., 2006, Nature 439:608-611; Ozbudak et al., 2002, Nature Genet 31:69-73), and on the other to play a fate determining role in systems as diverse as the sporulation-competence circuitry in B. subtilis (Suel et al., 2007, Science 315:1716-1919), mating pheromone response in yeast (Colman-Lerner et al., 2005, Nature 437:699-706), and HIV-1 latency (Weinberger et al., 2008, Nature Genet 40:466-470; Weinberger et al., 2005 Cell 122:169-182).
As discussed above, HIV-1 latency, a quiescently integrated viral state, has been identified as the leading barrier to completely eradicate the virus from infected individuals (Siliciano and Greene, 2011, Cold Spring Harb Perspect Med 1:a007096; Richman et al., 2009, Science 323:1304-1307). Upon infection of a cell, viral gene expression leads to either an active replication fate where the cell is hijacked of its resources to generate hundreds of viral progeny and ultimate cell death, or in rare instances, an inactive latent state where the provirus transcribes at undetectably low levels thereby evading anti-retroviral therapy. The HIV-1 promoter has high nucleosome occupancy including the stalling of RNA polymerase II after a nucleosome positioned at the transcriptional start site. This high nucleosome occupancy along with pol II stalling has been associated with higher gene expression noise in comparison to housekeeping promoters and generates diverse episodic transcriptional activity across the genome capable of modulation by signaling molecules (Singh et al., 2010, Biophys J 98:L32-L34; Dar et al., 2012, Proc Natl Acad Sci USA 109:17454-17459). Knockdown of the BAF nucleosome remodeling complex of the HIV-1 promoter was recently shown to increase latent reactivation into an actively replicating state (Rafati et al., 2011, Plos Biology 9:e1001206, 1-20). Together these observations support HIV-1 latency as a strong and clinically relevant phenotypic candidate for noise drug screening.
Applicants herewith provide compositions and methods useful for the elimination of latent HIV-1 reservoirs that persist despite HAART. The present invention is based, in part, on the Applicants' unexpected and surprising finding that noise modulating compounds identified by Applicants herein can synergize with an activator of latent HIV-1 expression, such as prostratin, to reactivate a latent HIV-1 reservoir.